Lipogenesis inhibition by certain esters of substituted benzodioxincarboxylic acids

ABSTRACT

Use as lipogenesis inhibitors in mammals of alkyl esters of certain substituted 2,3-dihydro-1,4-benzodioxincarboxylic acids.

This application is a continuation-in-part of application Ser. No.951,535, filed on Oct. 16, 1978, now abandoned.

DESCRIPTION OF THE INVENTION

It has been found that lipogenesis in mammals is inhibited by alkylesters of certain substituted 2,3-dihydro-benzodioxin-2-carboxylicacids, of the formula ##STR1## wherein "alkyl" is straight-chain orbranched-chain alkyl of from one to four carbon atoms, and R is a moietywhich is one of: formyl, acetyl, (hydroxyimino)methyl,1-(hydroxyimino)ethyl, (methoxyimino)-methyl, 1-(methoxyimino)ethyl,benzoyl, acetamido, amino, benzamido, methylsulfonylamino, and2-methyl-4-thiazolyl.

In Formula I, the dotted lines from the moiety R to the carbon atoms inthe ring are intended to indicate that the moiety can be bonded to thecarbon atom at the 6-position, or a carbon atom at the 7-position, ofthe ring structure.

Methods by which the compounds of this invention can be preparedordinarily produce mixtures of the 6-substituted and 7-substitutedspecies. Such mixtures, as well as deliberate mixtures, of the 6- and7-substituted species, as well as each of the individual 6- and7-substituted species, are contemplated in this invention.

Chirality exists in the compounds of Formula I due to the asymmetricstructural configuration at the 2-position of the2,3-dihydro-1,4-benzodioxin ring. As a result, each of these compoundsexists in the forms of two optical isomers. The individual isomers havenot been separated, so that their respective activity as lipogenesisinhibitors has not been determined. The invention contemplates each ofthe individual active isomers, as well as racemic, and other mixturesthereof.

Typical, exemplary, individual species of the compounds of Formula I,and their preparation, are described in actual examples set outhereinafter. Other typical, exemplary, individual species are thosebelow wherein, referring to Formula (I), "alkyl" and the moiety R, andits position on the ring structure, are as follows:

    ______________________________________                                        "alkyl"           R                                                           ______________________________________                                        ethyl             7-formyl                                                    ethyl             7-(hydroxyimino)methyl                                      methyl            7-(methoxyimino)methyl                                      ethyl             7-(1-methoxyimino)ethyl)                                    ethyl             7-benzoyl                                                   ethyl             7-acetamido                                                 methyl            7-acetyl                                                    n-propyl          7-acetyl                                                    isopropyl         7-acetyl                                                    l-methylpropyl    7-acetyl                                                    ethyl             7-amino                                                     ethyl             7-benzamido                                                 ethyl             7-methylsulfonylamino                                       ______________________________________                                    

and the 6-position counterparts of these species.

The compounds of Formula I can be prepared by the following procedures.

In each of these examples, the identities of the products, and theintermediates involved, were confirmed by appropriate elemental andspectral analyses.

EXAMPLE 1 Ethyl 7(and6)-acetyl-2,3-dihydro-1,4-benzodioxin-2-carboxylate (1)

400 ml of a mixture of 1:8 v/v glacial acetic acid in water was added intwo portions to a stirred mixture of 35 g of 4-(chloroacetyl)-catecholin 300 ml of ethanol. At 10 minute intervals, four 10 g portions of 90%zinc dust were added, the mixture being held below about 45° C. Themixture then was stirred for 2 hours at 30°-40° C. and allowed to standover a weekend. The liquid phase was decanted and stripped to a quarterof its volume under reduced pressure. The residue was extracted withether. The ether extract was washed with sodium bicarbonate solution.The solvent was evaporated. The solid residue was recrystallized fromtoluene to give 4-acetylcatechol (1A), mp: 118°-119° C.

150 g of ethyl 2,3-dibromopropionate was added over a 3-hour period to astirred slurry of 96 g of 1A 234 g of potassium carbonate and 600 ml ofacetone. The temperature of the mixture rose from 23° C. to 40° C. Themixture then was refluxed (55° C.) for 19 hours, and filtered. Theacetone was evaporated from the filtrate under reduced pressure. Theresidue was distilled to give 1, as a colorless liquid, bp: 165°-166°C., 0.1 Torr.

EXAMPLE 2 Ethyl 2,3-dihydro-7(and6)-(1-(hydroxyimino)ethyl)-1,4-benzodioxin-2-carboxylate (2)

A mixture of 18 g of 1, 75 ml of methanol, 5.2 g of hydroxylaminehydrochloride, 50 ml of water and 4.0 g of sodium carbonate was heatedon a steam bath for 35 minutes and allowed to stand overnight. The solidwhich formed was separated and recrystallized from benzene to give 2,mp: 111°-113° C.

EXAMPLE 3 Ethyl2,3-dihydro-7-(1-(methoxyimino)ethyl)-1,4-benzodioxin-2-carboxylate (3)

22.8 g of 1 was dissolved in 100 ml of ethanol. A solution of 7.94 g ofmethoxylamine hydrochloride and 5.5 g of sodium carbonate in 75 ml ofwater was added. The mixture was heated and stirred at refluxtemperature for 4 hours, then was allowed to cool and held at roomtemperature over a weekend. The mixture was diluted with 50 ml of waterand the solvents were evaporated. The residue was extracted with ether.The extract was dried (MgSO₄) and filtered. The solvent was evaporatedfrom the filtrate and the residue was chromatographed over silica gel,using a 1:1 v/v mixture of ether and hexane as eluent. The majorabsorption band was worked up to give an oil, which was distilled in aKugelrohr apparatus to give (3), b.p.: 136°-138° C. (0.12 Torr.).

EXAMPLE 4 Ethyl 6(and7)-(acetylamino)-2,3-dihydro-1,4-benzodioxin-2-carboxylate (4)

68 g of phosphorus pentachloride was added in portions over a 15-minuteperiod to a stirred solution of 50 g of 2 in 1000 ml of chloroform. Themixture was refluxed for 2 hours, allowed to cool while being stirred,then, with stirring, was poured into 1000 ml of ice water. The organicphase was separated, washed with water, then with 10% sodium bicarbonatesolution, dried (MgSO₄) and filtered. The solvent was evaporated fromthe filtrate under reduced pressure. The residue was triturated with 60ml of ethanol. The mixture was stored in a freezer overnight. The solidwhich formed was collected and washed with ethanol, then ether, anddried under reduced pressure. The product was dissolved in 100 ml of hotethanol; the solution was filtered through charcoal and chilled in afreezer over a weekend, and filtered. The solid was refluxed in 200 mlof ether for 1/2 hour and the resulting mixture was filtered. The solidwas chromatographed over silica gel using chloroform as eluent. Theappropriate fractions were extracted with chloroform and the solvent wasevaporated from the extract under reduced pressure. The residue wasagain chromatographed over silica gel, using chloroform as eluent. Theappropriate fractions were extracted with chloroform and the solvent wasevaporated from the extract under reduced pressure. The residue wasdissolved in 90 ml of hot ethanol, the solution was filtered throughcharcoal and chilled in a freezer overnight. The solid was collected,dissolved in hot ethanol; the solution was filtered through charcoal andchilled in a freezer. The solid was collected, ground and dried to give4, as a solid, mp: 146°-147° C.

EXAMPLE 5 Ethyl 7-benzoyl-2,3-dihydro-1,4-benzodioxin-2-carboxylate (5)

A solution of 110.1 g of catechol and 202 g of triethylamine in 1000 mlof tetrahydrofuran was added drop-by-drop to a stirred solution of 295.2g of benzoyl chloride in 1000 ml of tetrahydrofuran, at about 10° C. Thestirred mixture then was allowed to warm to room temperature and stirredfor 2 hours. The mixture was filtered and the solvent was evaporatedfrom the filtrate under reduced pressure (40° C.). The residue wassuspended in 500 ml of ligroin and stored overnight, then filtered. Thesolid was washed with petroleum ether and dried at room temperatureunder reduced pressure, to give catechol dibenzoate (5A), as a yellowcrystalline solid.

56 g of aluminum chloride was added to a stirred solution of 65 g of 5Ain 180 ml of nitrobenzene and the suspension was refluxed for 5 hours,then stored overnight. Then 800 ml of ice water was added, followed by25 ml of concentrated hydrochloric acid. The nitrobenzene was removed bysteam distillation. The aqueous phase was decanted and 333 ml of 3 Nsodium hydroxide solution was added to the residue. The mixture wasrefluxed for 3 hours. Concentrated hydrochloric acid was added to thestirred, cooled mixture until the pH of the mixture was about 1. Themixture again was refluxed, filtered through charcoal and the filtratewas placed in a refrigerator. The solid was collected, dried underreduced pressure (80° C.), and recrystallized from 350 ml of hot waterto give 3,4-dihydroxybenzophenone (5B).

11.63 ml of ethyl 2,3-dibromopropionate was added drop-by-drop over a15-minute period to a mixture of 15.9 g of 5B, 58.5 g of anhydrouspotassium carbonate and 250 ml of acetone. The stirred mixture wasrefluxed for 7 hours and stored overnight. The mixture was filtered andthe solvent was evaporated from the filtrate under reduced pressure (40°C.). The residue was suspended in 600 ml of ether and the mixture waswashed with water, then with sodium bicarbonate solution, then dried(MgSO₄). The solvent was evaporated under reduced pressure. The residuewas chromatographed over silica gel using chloroform as eluent. Workupwith ether and removal of the solvent gave a residue which was suspendedin ethanol and placed in a freezer overnight. The solid was collected,washed with ethanol and dried to give 5, mp: 70°-71° C.

EXAMPLE 6 Ethyl 6(and7)-(2'-methyl-4'-thiazolyl)-2,3-dihydro-1,4-benzodioxin-2-carboxylate(6)

A solution of 56.0 g of alpha-chloro-3',4'-dihydroxyacetophenone in 400ml of 1,2-dimethoxyethane was added drop-by-drop to a solution of 23.0 gof thioacetamide in 300 ml of 1,2-dimethoxyethane. The mixture washeated and refluxed for 20 hours. The precipitate which formed wascollected and dried. It was suspended in water and dilute (10%) sodiumhydroxide solution was added until the pH of the mixture was about 6.The precipitate was collected and dried to give4-(2'-methyl-4'-thiazolyl)catechol, (6A).

40.9 g of ethyl 2,3-dibromopropionate was added drop-by-drop to amixture of 31.1 g of 6A, 53.9 g of anhydrous potassium carbonate, and750 ml of acetone, at reflux. The resulting mixture was heated at refluxfor 18 hours, then was filtered. The filtrate was concentrated, theresidue dissolved in 500 ml of ether and the resulting mixture wasfiltered. The filtrate was washed sequentially with water, 5% solutionhydroxide solution, and water. The ether phase was separated, dried(MgSO₄) and concentrated. The resulting oil was chromatographed onsilica gel, using a 1:1 v/v mixture of hexane and ether as eluent. Thefraction containing the product was extracted with ether, and the etherwas evaporated under reduced pressure to give an oil. The oil wasdistilled to give 6, bp: 168°-172° C. (0.15 Torr.).

The individual species wherein R is formyl can be prepared from3,4-dihydroxybenzaldehyde according to the procedures described inExample 1.

The species wherein R is amino can be prepared by hydrolysis of thespecies wherein R is acetamido. The species wherein R ismethylsulfonylamino can be prepared by treating the species wherein R isamino with methanesulfonyl chloride. The species wherein R is benzamidocan be prepared by treating the species wherein R is amino with benzoylchloride.

Esters of Formula I have been found to inhibit lipogenesis in tissues ofmammals. The manner in which they cause this effect is not known withcertainty; it is believed that they interfere with the synthesis offatty acids in the tissues. Their effectiveness for this purpose hasbeen ascertained by immersing samples of swine adipose tissue in aliquid medium containing radioactive glucose and the test chemical for aperiod of time, then isolating the lipid from the treated tissue anddetermining the incorporation of the radioactive carbon into lipid bymeans of scintillation counting techniques. These tests were conductedin swine adipose tissues because in swine, the primary site oflipogenesis--i.e., fatty acid synthesis--appears to be adipose tissue.

Described in more detail, tests were conducted according to thefollowing general procedure:

150 milligrams of slices of swine adipose tissue were incubated at 37°C. for 2 hours with shaking in 3 milliliters of Krebs-Ringer bicarbonatesolution containing one-half the normal calcium ion concentration, 60micromoles of glucose, 0.5 micro-Curie of glucose-U¹⁴ C, and 300microunits of insulin, and 5% dimethyl sulfoxide (DMSO). The testcompounds were added as a solution or suspension in DMSO and werepresent at a concentration of 100 micrograms per milliliter of theincubation mixture.

The incubation was terminated by addition of 0.25 milliliter of 1 Nsulfuric acid. The resulting mixture was extracted with a total of 25milliliters of chloroform/methanol (2:1 v/v). The extracts were washedaccording to Folch et al. (J. Biol. Chem. 226, 497-509, (1957)), airdried, and counted in a liquid scintillation counter with 15 millilitersof counting fluid (two parts toluene containing 0.4% w/v New EnglandNuclear Omnifluor/1 part Triton X-100). The tests were conducted intriplicate and were accompanied by control tests in which allingredients, proportions and conditions were the same except that notest compound was included. From the data obtained were calculated thepercent inhibition of lipid synthesis by the test compounds in eachcase. The data obtained from the tests are set out in Table 1, as thepercent inhibition of lipogenesis compared to the results obtained inthe control tests wherein only the test compound was omitted.

                  Table I                                                         ______________________________________                                        Compound No.       Percent Inhibition                                         ______________________________________                                        1                  89                                                         2                  94                                                         3                  52                                                         4                  96                                                         5                  69                                                         6                  79                                                         ______________________________________                                    

The esters of Formula I can be used to control lipogenesis in mammalssuch as, for example, pets, animals in a zoo, livestock, furbearinganimals and domestic animals, including, but not limited to dogs, cats,mink, sheep, goats, swine, cattle, horses, mules and donkeys. The effectis obtained by administering an effective amount of one or a mixture oftwo or more of the esters orally or parenterally to the animal. They maybe administered as such, or as an active ingredient of a conventionalpharmaceutical formulation. They may be administered orally by anyconvenient means. Thus, they may be orally administered as a drench, byintubation, in the animal's food and water, in a food supplement or in aformulation expressly designed for administration of the drug. Suitableformulations include solutions, suspensions, dispersions, emulsions,tablets, boluses, powders, granules, capsules, syrups and elixirs. Forparental administration, they may be in the form of a solution,suspension, dispersion or emulsion. They can be administered in the formof an implant or other controlled sustained release formulation. Inertcarriers, such as one or more of water, edible oil, gelatin, lactose,starch, magnesium sterate, talc or vegetable gum can be used. The dosageof the ester needed to inhibit lipogenesis will depend upon theparticular ester used, and the particular animal being treated. However,in general, satisfactory results are obtained when the esters areadministered in a dosage of from about 1 to about 400 milligrams perkilogram of the animal's body weight. The ester can be administered in asingle dose or in a series of doses in the same day, or over a period ofdays. For any particular animal, a specific dosage regimen should beadjusted according to the individual need, the particular ester(s) usedas the inhibitor, and the professional judgment of the personadministering or supervising the administration of the inhibitor. It isto be understood that the dosages set forth herein are exemplary only,and that they do not, to any extent, limit the scope or practice of theinvention.

I claim as my invention:
 1. A method of inhibiting lipogenesis in amammal, which comprises administering, to a mammal in need of suchtreatment, orally or parenterally a lipogenesis inhibiting amount of acompound of the formula ##STR2## wherein "alkyl" is alkyl of from one tofour carbon atoms, and R is a moiety which is one of: formyl, acetyl,benzoyl, acetamido, (hydroxyimino)methyl, 1-(hydroxyimino)ethyl,(methoxyimino)methyl, 1-(methoxyimino)ethyl, amino, benzamido,methylsulfonylamino, and 2-methyl-4-thiazolyl.